The minimal upstream regulatory region of the Hspa2 gene required to determine the germ cell-type and stage-specific expression of the gene. Different Hspa2 gene promoter fragments were ligated to the Lacz reporter gene and beta-galactosidase expression determined in the testes of transgenic mice. It was found that sequences within 604 bp of the translation start site are required for correct expression. This region was examined further with in vitro methods. Footprint analysis identified two domains protected from DNase digestion by germ cell nuclear proteins, referred to as box 1 (between bp -555 and -503) and box 2 (between bp -346 and -335). These domains contain clusters of transcription factor binding motifs. Gel shift and super-shift analyses indicated that several known transcription factors and unknown proteins present in germ cell nuclei bind to specific sequences in these regions. HSPA2 is synthesized during the meiotic phase of male germ cell development and we hypothesized that it is a chaperone for proteins involved in meiosis. This was confirmed using the gene knockout approach. Disruption of the Hspa2 gene resulted in developmental arrest and apoptosis of pachytene spermatocytes at the G2/M-phase transition of meiosis I. We subsequently found that HSPA2 serves as a chaperone for CDC2 and is required for assembly of the CDC2/cyclin B1 meiosis promoting complex. More recent studies identified an unexpected role for HSPA2, a tight association with the major spermatid DNA-packaging proteins, transition proteins 1 and 2. This suggests that HSPA2 also serves as a chaperone for the transition proteins and participates in the process of nuclear condensation that occurs in spermtids. [unreadable] [unreadable] The HSPA1L protein is present only in spermatids, during the post-meiotic phase of male germ cell development. By analogy with HSPA2, we hypothesized that HSP1L is a chaperone for unique proteins involved in post-meiotic germ cell development or sperm function. Male Hsp1l knockout mice have normal fertility and there are no apparent changes in testis morphology or in sperm numbers but sperm from Hspa1l-/- mice incubated for longer than 30 minutes in vitro became immotile, while those from wild-type retained motility for several hours. ATP levels of sperm from Hspa1l-/- mice is about 1% of that in wild-type sperm and cAMP levels are about half. Variability in the phenotype was suspected to be due to the mixed genetic background of the mice and backcrossing studies are currently underway to test this hypothesis.